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Summary The plant alkaloid camptothecin inhibits interferon production induced by Newcastle disease virus (NDV) or ultraviolet-irradiated NDV in chick and human cells, and by Sindbis virus in chick cells. It has no effect on interferon production induced by poly(rI).poly(rC) in chick and human cells. No effect of camptothecin could be detected on the multiplication of NDV, and it is concluded that the inhibition reflects a difference between interferon induction by viruses and by polynucleotides.

Twenty-four children with cancer and localized herpes zoster were randomized to receive either topical polyinosinic acid-polycytidylic acid [poly(I:C)] or a saline placebo. Solutions containing 1.0 or 2.5 mg of drug per ml were applied to the lesions every 3 h, seven times a day for 7 consecutive days. Serial, standardized color photographs were used to evaluate the progression of lesions and maximum percentage of dermatome involvement. In patients receiving poly(I:C), the median days of new lesions after therapy was 2.0 days as opposed to 3.5 days in placebo-treated patients. This difference was not statistically significant. Moreover, the total median days of lesions and percentage of dermatome involvement were similar in both groups, as were complications. Neither the concentration nor the total dose of drug was related to the outcome of infection. We conclude that topical poly(I:C), as used in this study, was of no benefit in treatment of localized herpes zoster.

Two lysosomotrophic drugs, neutral red and chloroquine, enhance polyinosinic:polycytidylic acid-induced interferon production by a strain of diploid human fibroblasts (FS-4). Treatment of cells with neutral red or chloroquine between 2.5 and 3.5 h after induction increases interferon yields 16- to 64- and 4- to 16-fold, respectively, in the subsequent 20.5 h. The two drugs inhibit the rates of protein degradation and of RNA and protein synthesis. In addition, neutral red is a very potent inhibitor of uridine transport into cells. Normalized dose-effect curves show that interferon superinduction is correlated with the inhibition of macromolecular synthesis, but not with that of protein degradation. Treatment of cells with chloroquine at low concentration (25 mug/ml) for a prolonged period of time (24 h) caused approximately 40% reduction in the rate of protein degradation. The usual rapid shutoff of interferon production and the effectiveness of effectiveness of actinomycin D superinduction are not altered by this treatment. This strongly suggests that inhibition of intralysosomal protein degradation does not significantly contribute to interferon superinduction. Degradation of the rapidly and the slowly turning over proteins was unaffected by actinomycin D under conditions of treatment known to enhance interferon production. Treatment with cycloheximide (5 or 50 mug/ml for 5 h) inhibited the rate of degradation of the rapidly turning over component by 10% and the slow component by 30-40%, which suggests that the two components turn over by distinct cellular mechanisms.

Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I)·poly(C)] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(I)·poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer. Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about 10–15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(I)·poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(I)·poly(C)-induced interferon response. Poly(I)·poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(I)·poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.

Viral infection by vesicular stomatitis virus (VSV) as well as cellular protection against it were studied in cultured human diploid fibroblast cells (WI38 strain) of varying senescence (passage) level. Full protection against viral infection can be achieved by pretreatment of cells with an interferon inducer (complex of polycytidylate and polyinosinate) or by pretreatment with concentrates of interferon itself. The late passages (over 45) require higher concentration of both agents than the medium passages (25–45); a complete failure of protective mechanisms occurs only close to cellular death. Furthermore, effects of confluency and of duration of induction impulse were studied. WI38 cells are sensitive to VSV infection through their entire life span; however, during the last passage before their death by senescence, VSV replication is significantly decreased. It is concluded that even in relatively senescent cells (a) protection mechanisms which were not used previously can be activated, (b) synthesizing machinery necessary for efficient support of viral replication is sustained, and (c) release of lysosomal enzymes ending the viral replication does not begin sooner than with cells of earlier passage level.

SummaryThe inactivation of poly(I)·poly(C)-induced and virus-induced human fibroblast interferon, virus-induced human leukocyte interferon, poly(I)·poly (C)-induced and virus-induced mouse tissue culture interferon and virus-induced mouse serum interferon by shaking and by guani-dine hydrochloride was studied. Only human leukocyte interferon was completely resistant to these treatments. All other interferons examined were largely inactivated, but a small amount of each preparation was resistant to inactivation. The residual activity from either treatment was completely resistant to the other.Inactivation by both shaking and guani-dine hydrochloride was prevented in acid (pH 2 and 3.5) conditions. Possible explanations for these observations are considered.

The synthesis of viral complementary RNA by vesicular stomatitis virus in permissive cells can be distinguished operationally into two phases, primary transcription, which can be observed in cycloheximide treated cells, and secondary transcription, which represents the further synthesis of viral complementary RNA obtained in untreated cells. Pretreatment of mouse L cells or chicken embryo fibroblasts with the homologous interferon and, for chicken embryo fibroblast cells, poly(rI):poly(rC), inhibits the production of infectious virus and reduces the synthesis of viral complementary RNA to levels comparable to that obtained during primary transcription. Treatment of mouse L cells with interferon plus cycloheximide also gives levels of vesicular stomatitis viral complementary RNA synthesis comparable to that observed with the cycloheximide treatment alone. Similar results have been obtained for interferontreated cells subsequently infected with influenza virus (strain WSN). The results are interpreted as indicating that inhibition of virus development by interferon does not act at the level of primary transcription but rather at an intermediate step between primary and secondary transcription, such as viral protein synthesis.

Herpetic keratoconjunctivitis, induced in rabbit eyes, was experimentally treated with poly-inosinic and poly-cytidilic acid (poly I : C), idoxuridine (IDU) and a combination thereof. It appeared that IDU 0·1% four times daily and poly I:C 1 mg/ml twice daily combined are more effective than either IDU four times daily or poly I:C twice or eight times daily alone. The best results were obtained with the combination of IDU and poly I:C, when the poly I:C was freshly prepared every day. Further observations were that treatment with poly I:C alone was more effective when started on the first or second day after viral inoculation of the rabbit eyes than when started on the third or fourth day.

The in vitro chemotactic response of peritoneal macrophages from mice infected intraperitoneally (ip) with Mycobacterium bovis, strain BCG, was greater than the response of macrophages from uninfected mice in terms of rate, magnitude, and sensitivity. This increase in chemotactic responsiveness induced after a single ip injection of BCG was apparent in macrophages harvested 15 hr later, became maximal by 3 wk, and persisted through 6 wk. Peritoneal macrophages from mice treated with one of several macrophage activation agents (endotoxin, polynucleotides, or concanavalin A) had chemotactic responses to both lymphocyte and complement-derived chemotactic factors comparable to that seen with BCG-activated macrophages. Macrophages from peritoneal exudates induced by oil or thioglycollate, however, were less responsive to chemotactic stimuli than were macrophages from untreated mice. Increased chemotactic responsiveness was a functional property of the activated macrophage.

L-929 cells have been cultivated for more than 6 months in the presence of interferon. These L-cells, referred to as L-int cells, were found to be more susceptible to the lytic properties of vesicular stomatitis virus (VSV) than normal L-929 cells. This phenomenon has been demonstrated by the production of clear plaques as opposed to cloudy plaques as well as the increased release of cell incorporated 3H-thymidine after VSV infection. The L-int cells were also signicantly more susceptible than L-929 cells to the cytotoxic properties of DEAE-dextran, protamine sulfate, and methylated albumin.This interferon-induced change to L-cells is not stable. Increased susceptibility to VSV lysis is lost after 4–6 cell generations in the absence of interferon, and increased susceptibility to DEAE-dextran is lost after 16–24 cell generations in the absence of interferon. Moreover, the increased sensitivity to VSV lysis and polycation cytotoxicity can be induced after overnight treatment of L-929 cells with interferon.

A comlex of polyriboinosinic-polyribocytidylic acid (poly I-poly C) with poly-L-lysine and carboxymethylcellulose has been prepared. This complex is five to 10 times as resistant to hydrolysis by primate serum as the parent poly I-poly C. It has a thermal denaturation temperature about 40 C higher than that of poly I-poly C. The complex induces significant levels of serum interferon in monkeys and chimpanzees under conditions in which poly I-poly C itself induces no interferon.

The synthesis of the enzyme, tyrosine aminotransferase, can be induced in rat hepatoma cells in cell culture by treatment with adrenal steroids. Partial inhibition of the induction requires exposure of the cells to 200–700 times higher concentrations of a partially purified rat interferon preparation than those required for a comparable inhibition of reovirus or vesicular stomatitis virus replication. These results reveal the selectivity of interferon action.

In terms of human disease, the arboviruses producing the greatest number of clinical cases of central nervous system illness in the United States are St. Louis encephalitis (SLE) virus, La Crosse (LAC) virus (the predominant pathogenic member of the California encephalitis virus group), and Western equine encephalitis (WEE) virus [1]. Although clinical illness due to LAC virus tends to be relatively mild, SLE in elderly persons and WEE, particularly in infants, can be highly lethal encephalitic processes [2, 3]. In previous reports, we have detailed the measurement of interferon in the brains of persons dying from SLE and WEE [4, 5]. In the case of SLE, the maximal concentration of interferon measured was equivalent to 75 international reference units (IRU)/g (wet weight) of tissue. In contrast, a concentration of interferon equivalent to 4,800 IRU was found in the brain of a person dying from WEE. These patients were all > 60 years of age. In vitro experiments with mononuclear cells from adult nonimmune volunteers confirmed that WEE

Thermal-denaturation profiles of helical polynucleotides have been measured in the presence of increasing concentrations of ethidium bromide. The poly(A)·poly(U) helix is strongly stabilized by binding of ethidium, to much the same extent as is DNA, but the stabilizing effect on poly(I)·poly(C) is much smaller. In the poly(A)·2poly(U) system the drug selectively destabilizes and eventually destroys the triple helix, leaving only the double-helix-to-coil transition.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.

Radical-induced copolymerization of 1-vinyluracil and maleic anhydride gave, after hydrolysis, a polymer containing a 1:1 monomer ratio of 1-vinyluracil-maleic acid. Gamma-Ray-induced copolymerization of 1-vinyluracil with acrylic acid gave a polymer with a ratio of 1:1.7. Similar treatment of 9-vinlyadenine and acrylic acid resulted in a polymer with a 1:3.2 ratio. These three compounds are potent stimulants of poly (uridylic acid) coded polyphenylalanine synthesis in an in vitro cell free system purified from Escherichia coli MRE 600. The double-stranded polymer, poly(inosinic acid)-poly(cytidylic acid), also stimulates polyphenylalanine synthesis in this assay.

A local antiviral effect can be observed when (poly rI)-(poly rC), bound to Visking discs by u.v. irradiation, is incubated with monolayers of human foreskin fibroblast cells. Radioactive labelling of cytosine residues in (poly rI)-(poly rC) with -125I, has provided a much more sensitive method for determining the fate of the insoluble (poly rI)-(poly rC) than has been available hitherto. The antiviral effect is not related to the amount of (poly rI)-(POLY RC) present on the insoluble support but rather to the amount of polynucleotide lost from the support during incubation. Treatment of (poly rI)-(poly rC) which had been bound to cyanogen bromide-activated Sepharose with eigher dilute alkali or pancreatic ribonuclease released virtually all the polynucleotide. A small amount of (poly rI)-(poly rC) is released from the insoluble matrix in the presence of serum-free Minimum Eagle's Medium.

Treatment of Friend virus (FV)-infected mice, 3 days after FV inoculation, with statolon, an extract of the mold Penicillium stoloniferum, induces interferon and restores immunocompetence to viral and nonviral antigens such as sheep erythrocytes. Clinical remissions are established in 20 to 70% of the infected mice. Cholorite-oxidized oxyamylose administered intraperitoneally 24 h before, 3 h before, or 3 h after statolon enhanced interferon production, but the increased number of mice protected against FV disease was more closely related to the associated enhanced synthesis of FV cytotoxic antibody. The prolonged selective immunodepression to intraperitoneal sheep erythrocytes after intraperitoneal administration of chlorite-oxidized oxyamylose-statolon appeared to be related to a stimulation in number and erythrocyte-phagocytic capacity of peritoneal macrophages. The marked activation of macrophages in FV leukemic mice after such treatment may also have contributed to the enhanced FV leukemosuppressive effects of chlorite-oxidized oxyamylose-statolon.

Membrane fractions from chick embryo cells manifesting viral interference mediated by interferon or poly(I)-poly(C) contain high levels of an alkaline ribonuclease. Enhanced RNase activity is not observed when inhibitors of cell protein or RNA synthesis are present during interferon treatment, or when heterologous interferon is used. The RNase associated with comparable membrane fractions from cells treated with mock-interferon is about 1/10 as active, and shows qualitative differences. In principle, divergent views of interferon action may be reconciled to a common mode of action by postulating that viral interference results from a newly induced or activated RNase of cellular origin and proper specificity that acts to reduce the accumulation and functional capacity of newly synthesized viral RNAs, particularly mRNA. Previous data in support of interferon's acting to inhibit virion-derived transcription in vivo are now interpreted as demonstrating enhanced degradation of viral transcripts (mRNA).

Mild treatment of 3T3 cells with low concentrations of Clostridium perfringens neuraminidase and phospholipase C resulted in inhibition of interferon production. The enzymatic digestion did not affect the viability and plating efficiency of these cells. Treatment of the cells with concentrations of neuraminidase and phospholipase C, which caused complete suppression of the rI n · rC n response did not affect binding of the inducer to these cells. Thus, the antiviral response and cellular binding properties of rI n ·rC n can be dissociated. The rI n ·rC n inducible interferon production of neuraminidase- and phospholipase C-treated cells was restored upon prolonged incubation in media. The observed inhibition was not specific for 3T3 cell systems; human fibroblast cells responded similarly. Finally, radioactive DEAE-dextran was synthesized and its binding to 3T3 cells was studied under conditions which potentiate interferon induction. While the binding of rI n ·rC n is greatly enhanced in the presence of DEAE-dextran, the binding of DEAE-dextran was independent of the presence of rI n ·rC n .

Summary The acid-insoluble radioactivity associated with the cells following incubation of the cells with [3H]-labelled poly(I).poly(C) [poly(I).poly(C) * for 1 h has been analysed by sucrose gradient velocity ultracentrifuging. Intact (poly(I).poly(C) * was recovered from primary rabbit kidney (PRK) cells, partially degraded poly(I). poly(C) * was recovered from mouse L 929 cells and completely degraded material was recovered from HeLa and VERO cells. Priming of the cells with homologous interferon did not alter the sedimentation profile of cell-associated poly(I).poly(C) * in PRK, L 929 or HeLa cells.

Antibodies to viral double-stranded RNA (ds RNA) have been found in 40% of patients with systemic lupus erythematosus (SLE) and 14-5% of patients with rheumatoid arthritis. These antibodies were diagnostically more specific SLE than those directed against artificial polynucleotides, poly I:C and poly A:U. Although not disease specific, high titres of antibody to ds viral RNA were found almost exclusively in SLE. Serial studies failed to show that RNA antibody levels correlated with disease activity. Although of considerable interest in experimental studies on the pathogenesis of SLE, ds viral RNA antibodies are of little clinical significance in the management of SLE.

Complexing of polynucleotides to polycationic substances enhances their uptake by mouse peritoneal cells. DEAE dextran and protamine sulfate when complexed with poly(rI) · poly(rC) lost their ability to release histamine while the complexes become very active in their interferon inducing capacity. Increased concentrations of poly(rI) · poly(rC) in the complex with protamine sulfate resulted in increase of interferon production but in decrease of histamine liberation. As the cationic character of histamine liberators is not necessary for this process, results presented here suggest, that the cell membrane may play an important role in the interferon production triggered by polynucleotides.

Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes in vivo nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon. It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I-poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4-6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I-poly C. There was no significant difference in the binding rate of poly I-poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I-poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.

A murine model simulating human street rabies virus infection was used to evaluate the efficacy of polyriboinosinic-polyribocytidylic acid (poly I-poly C), three rabies vaccines, and combinations of these modes of therapy administered after exposure. One or two doses of 100 mug of poly I-poly C, injected into the same intramuscular site as the challenge virus, significantly reduced the mortality rate when therapy was initiated 3 hr after challenge; however, the same quantity of poly I-poly C injected into the opposite leg did not reduce the mortality rate. The muscle injected with poly I-poly C invariably contained four to eight times more interferon than a similar noninjected muscle from the same animal. Mice treated 3 hr after challenge with each of the three vaccines produced significant levels of antibody but were not protected, whereas treatment with combinations of poly I-poly C and vaccine resulted in significant protection. These results suggest that the combination of induction of local interferon and an immune response contributes to the protection of mice after exposure to street rabies virus infection.

The mechanism by which poly(I · C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I · C)], the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I · C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(I · C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (I · C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(I · C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell.

An antiserum was prepared in rabbits to the synthetic double-stranded ribonucleic acid (ds RNA) poly rI:rC. Using a liquid-phase radioimmunoassay, the antiserum cross-reacted with a natural ds RNA isolated from the cytoplasmic polyhedrosis virus of the silkworm, binding 95% of the RNA at a 1 : 20 serum dilution. Preliminary tests of the specificity of the antiserum showed that it did not bind single-stranded RNA (ss RNA) or deoxyribonucleic acid (DNA), but also revealed that the serum contained an enzyme activity which degraded ss RNA into acid-insoluble fragments. It was therefore possible that the failure to bind ss RNA resulted from the degradation of the antigen rather than from an absence of cross-reacting antibodies. However, when the serum ribonuclease activity was inhibited by macaloid, the antiserum still did not bind the ss RNA antigen. This demonstrated that the antibodies to ds RNA did not cross-react with ss RNA. The existence of serum enzymes capable of degrading nucleic acid antigens emphasizes the need for caution in assessing the specificity of such antisera.

Mouse spleen cells, cultured on surfaces coated with antigen-antibody complexes, are inhibited from responding to the B-cell mitogens, lipopolysaccharide, lipid A, Pneumococcal polysaccharide SIII, and poly I:C. The response to the T-cell mitogen, concanavalin A, is also substantially inhibited by immobilized antigen-antibody complexes, but specific inhibition of the response to phytohemagglutinin is minimal. Control experiments showed that immobilized complexes prepared from IgG F(ab')2 fragments and IgA antibodies (both of which fail to bind to Fc receptors when complexed to antigen) did not show significant inhibitory activity when compared with the inhibition observed with complexes prepared from whole IgG. Suspensions of antigen-antibody complexes prepared from the same antigen and intact IgG antibody did not inhibit mitogenesis. None of the mitogens used could be demonstrated to compete with the binding of aggregated immunoglobulin to the B-cell Fc receptor. It appears that the interaction of Fc receptor-bearing lymphocytes and/or macrophages with immobilized complexes prevents lymphocyte activation by mitogens. It is suggested that the mechanism(s) involved may be relevant to antibody feedback control of the humoral immune response.

Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in response to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes to vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation.

Summary Mild treatment of chick embryo and L cells with proteases resulted in decreased induction by poly(rI).poly(rC) of interferon and of antiviral resistance, as well as in reduced uptake of [3H]-poly(rI).poly(rC). The enzyme treatment of the cells neither significantly affected their sensitivity to exogenous interferon nor suppressed interferon induction by a virus inducer. The supernatant fluid from the trypsintreated cells contained a substance which complexed with poly(rI).poly(rC), resulting in inhibition, but not in irreversible inactivation, of its interferon-inducing ability. The induction was also inhibited when the cells were first incubated with the inducer and then treated with trypsin. Since trypsin presumably acts primarily on the cell surface, these results are interpreted to indicate that the binding of poly(rI).poly(rC) to cell surface sites and its persistence at the surface for a certain period of time plays an essential role in interferon induction.

The metabolic inhibitors, actinomycin D, cycloheximide, puromycin dihydrochloride, puromycin aminonucleoside, and p-fluorophenylalanine did not inhibit the release of leukocytic pyrogen whether endotoxin was preincubated with cells for 20 min at 37 degrees C before addition of inhibitor or inhibitor was preincubated with cells for 1 hr before addition of endotoxin. On the other hand, cortison inhibited release of pyrogen under both experimental conditions. Poly(I): poly(C) was not effective in inducing rabbit leukocytes to produce an endogenous pyrogen.

BL-20803, a low-molecular-weight compound, although able to elicit circulating interferon in the mouse, failed to protect cultured cell lines in vitro from infection by interferon-sensitive viruses. Of the tissues analyzed for interferon content after oral administration of the drug to mice, spleen and lung contained the largest amounts of the virus inhibitor. Spleen cells from such dosed animals when isolated into in vitro cultures elaborated small amounts of interferon into the culture medium. The time sequence of acquisition by spleen cells of the ability to produce interferon closely correlated with the kinetics of development of the circulating interferon response in the intact mouse. When spleen cells were separated on the basis of adherence or nonadherence to a plastic surface, the bulk of the interferon activity was found to be associated with the adherent cells. Upon exposure to BL-20803 in cell culture, adherent cells and, to a lesser extent, nonadherent cells from untreated mice were stimulated to produce interferon-like activity. The biological behavior of BL-20803 is shown to have striking similarities with that of the structurally different low-molecular-weight inducer tilorone hydrochloride.

Diminution of cerebral protein synthesis diminished the cerebral responses of mice to some neuroactive drugs, while an increase in synthesis increased the responses. Protein synthesis in whole brains (tested in vitro ) was diminished by giving living mice different inhibitors by different routes. The inhibitors tested (chloramphenicol, cycloheximide, and puromycin) diminished the behavioral responses of the mice to levodopa without affecting either its cerebral uptake or its conversion to dopamine. A diminution of the reactions of dopaminergic receptors was suggested by the diminished responses to the dopaminergic drug, apomorphine, while participation of cholinergic ones was suggested by experiments with oxotremorine. Proof that receptors had been specifically involved was secured on homogenized caudate nuclei from chloramphenicol-treated mice, in which the dopamine-activated production of cyclic AMP was markedly diminished. A stimulator of cerebral protein synthesis, the artificial double-stranded RNA, poly(I)·poly(C), increased the behavioral responses to these three drugs while it increased the dopamine-activated production of cyclic AMP. Since all these experimental increases or decreases in the responses to drugs required the lapse of only a few hours, proteins with rapid turnover rates must be critical in the activation of several kinds of cerebral receptors.

The effect of emetine on the in vivo production of interferon by Swiss mice stimulated with poly-i: poly-C, was studied. 100 mug of emetine per mouse yielded a 16-fold increase in interferon production. Maximal production of interferon was achieved 2 h after the simultaneous injection of emetine and poly-I: poly-C.

Poly(I).poly(C) covalently coupled to a matrix by one point fixation through its 3′ terminal stimulated both antiviral activity and interferon production in primary rabbit kidney (PRK) cells. This effect could not be accounted for by free polynucleotide released from the matrix into the medium. Penetration of the polynucleotide into the cells does not appear to be necessary for interferon production. A limited amount of matrix-bound poly(I).poly(C) was associated with the cells. Since it was sensitive to extraneous ribonuclease treatment, this poly(I).poly(C) was believed to be localized at the cell surface. Preliminary findings suggest that the binding of the polynucleotide to the cell is not directly proportional to the amount of interferon induced.

Summary Interferon-treated L cells exhibit a specific enhanced susceptibility to the cytotoxic and interferon inducing activities of double-stranded RNAs such as poly(I).poly(C). These activities remained closely linked through widely varying assay conditions, involving, for example, different time and dosage schedules of poly(I).poly(C), suggesting that there is at least one common step in the mechanisms leading to interferon formation and toxicity in interferon-primed cells exposed to poly(I).poly(C). However, some procedures such as addition of metabolic inhibitors (actinomycin D, cycloheximide) and repeated administration of poly(I).poly(C) suppressed the interferon inducing capacity of poly(I).poly(C) without a concomitant decrease of toxicity. Other procedures such as brief treatment of the cells with interferon or DEAE-dextran permitted full expression of the interferon inducing activity of poly(I).poly(C) without any sign of toxicity. The latter results suggest that the mechanisms underlying interferon production and toxicity of poly(I).poly(C) in interferon-treated L cells diverge from a certain point onward.

Cellular immunity of 16 patients with systemic lupus erythematosus (SLE), as measured by the ability of their lymphocytes to proliferate in vitro following stimulation with mitogens and bacterial antigens, and by the ability of their polymorphonuclear neutrophils to reduce nitrobluetetrazolium dye, both spontaneously and upon stimulation, was found to be unimpaired by comparison with cells of 12 normal individuals. The relative frequency of responders to stimulation with human collagen and with several nuclear antigens, used over a wide range of concentrations and evaluated as a group, was significantly higher among patients with SLE. Antinuclear antibodies specific for DNA, RNA, or both, were found in all patients with SLE, but did not reflect the pattern of sensitization of lymphocytes to these antigens as measured by lymphocyte transformation.

Sera from patients with systemic lupus erythematosus were fractionated by sucrose density gradient ultracentrifugation. Individual fractions were assayed for immunoglobulin and for antibody activity to human DNA, to reovirus RNA, and to a DNA:RNA hybrid (deoxythymidylic·riboadenylic acid, dT·rA). Antibodies to dT·rA were distributed in two peaks corresponding to 19S IgM and 7S IgG. Antibodies to DNA were predominantly IgG, whereas antibodies to RNA showed a more variable distribution. IgM antibodies to RNA were quite prominent and in some patients were the only anti-RNA antibodies detected. These results are discussed in relation to genetic and antigenic factors which determine the immunoglobulin class of anti-nucleic acid antibodies.

Lead acetate was administered continuously in the drinking water to CD-1 male mice beginning at 4 weeks of age. An LD(10-20) of the lytic viruses or 300 plaque-forming units of RLV was inoculated intrapertioneally at 6 weeks of age. Lead increased the response of the mice to all classes of viruses against which it was tested: an RNA picornavirus-encephalomyocarditis (EMCV), a DNA herpesvirus-pseudoribies, an RNA leukemia-virus-Rauscher leukemia (RLV), an RNA arbovirus B-St. Louis encephalitis, and an RNA arbovirus A-western encephalitis. Most studies were performed between lead and EMCV. Increases in EMCV mortality in lead treated mice over controls ranged from 2x at a lead level of 0.004M to 7x (100% mortality) at a 0.1M lead level. Splenomegaly with spleens 800 to 1100 mg in weight containing high titers of RLV occurred in lead (0.03M)-treated mice 3 and 6 weeks after RLV inoculation; spleens or RLV controls were normal in weight (200 mg) and were free of virus. Lead did not reduce the protective effect of mouse interferon (IF) against the lethal action of EMCV, but it did repress the EMCV antiviral effect of poly I/poly C (PIC) and of Newcastle disease virus (NDV) against EMCV mortality. These data indicate several new facts concerning adverse effects lead may have on an animal: (1) lead aggravates viral disease, most likely in part, through reduced IF synthesis; (2) lead represses the anti-EMCV protective effects of both PIC and of NDV, which, in other reports, were shown to induce IF in radioresistant macrophages (PIC) or in radiosensitive lymphocytes (NDV); (3) lead may then be said to repress IF induction in two kinds of cells; (4) however, lead does not inhibit IF action.

Treatment of diploid human foreskin cell cultures with either 2-deoxy- d -glucose or d -glucosamine resulted in dose-dependent inhibition of interferon production induced with polyinosinate-polycytidylate [poly(I) · poly(C)]. This inhibitory action was readily demonstrable even if the addition of the inhibitor was delayed until 1.5 hr after induction. The appearance of intracellular interferon activity was also inhibited, suggesting that synthesis of biologically active interferon, and not its secretion from the cell, was the main target of the inhibitory action. The inhibitory action of 2-deoxy- d -glucose on interferon production was completely reversible by d -mannose while the action of d -glucosamine was only slightly affected by the addition of mannose. Interferon produced in the presence of a partially inhibitory concentration of 2-deoxy- d -glucose was more heat-labile than control interferon. Residual active interferon made in the presence of d -glucosamine was less efficiently neutralized by anti-interferon antibody than control interferon. The results are compatible with the idea that the two inhibitors suppress the production of biologically active interferon by virtue of their interfering with the proper glycosylation of the molecule.

SummaryA single dose of the synthetic double-stranded polynucleotide poly I·C inoculated intraperitoneally into mice 12 to 48 hr before challenge with coxsackievirus B-3 resulted in almost complete protection from virus-induced myocarditis. Protection was related to the presence of high titers of circulating IF in the serum. Significant protection was also obtained even when poly I·C was given 24 hr after challenge with coxsackievirus.

SummaryFunctional interaction between histone and RNA was studied using Nelson mouse ascites cells. Liver RNA was capable of stimulating RNA synthesis and arginine-rich histone inhibited it. The potency of low molecular weight fraction was superior to the high and whole RNAs, but inferior to the nucleolar RNA. Poly I-C was unable to lift the RNA synthesis to a level above the controls.Intracellular distribution of 125I-histone was traced by high resolution autoradiography. The predominant nuclear uptake of histone coupled with early work on RNA uptake provided evidence for the in situ action of histone and RNA. The RNA-stimulated RNA synthesis was sensitive to actinomycin D. From studies on the action of RNA versus histone, it was concluded that the action of RNA was mediated by arginine-rich histone.Fig. 10. The response of the histone pretreated cells (H) to RNA (HR) and RNAse-digested RNA (RNAse). The concentration of histone and RNA is same as Fig. 9 (3H-uridine triphosphate as precursor).

Antibodies against a single minor base, inosine, have been prepared by immunizing rabbits with a conjugate of inosine and bovine serum albumin. The antisera obtained were fairly specific for inosine, only guanosine among the major nucleosides being capable of cross-reacting with them. By adsorption with guanosine-bovine serum albumin, antibodies with a specificity directed almost exclusively against inosine were obtained. The anti-inosine sera were able to cross-precipitate with polyinosinic acid if the latter was in single-stranded form. Inosine residues in polyinosinic acid paired with cytidine residues of polycytidylic acid, forming a double-stranded helix, were unable to react with the anti-inosine antibodies. The triple-stranded form of polyinosinic acid under stable conditions was also unable to form an immune precipitate with anti-inosine antibodies.

Since viruses depend on cellular enzymes for their synthesis, they are less amenable than more complex organisms to selective inhibition. However, our knowledge of viral-specific intracellular events has increased and the feasibility of viral chemotherapy has become more firmly established at least in selected situations. For example, idoxuridine, methisazone, and amantadine represent three separate chemical classes of agents generally considered to be efficacious in the treatment of viral infections in man. The use of idoxuridine against herpes keratitis in man is well established; its clinical utility for dermal herpetic infections is less clear. Other unnatural nucleosides such as Ara-C and Ara-A also exhibit antiviral activity in various experimental infections. Methisazone has been used to prevent smallpox and alastrim in clinical contacts; it has also been used with apparent success in the treatment of vaccinia gangrenosa and eczema vaccinatum. Other thiosemicarbazones also possess similar intrinsic antiviral activity. Amantadine inhibits certain strains of influenza. Prophylaxis has been achieved in challenge studies and field trials against influenza A2. From more limited studies it also appears that this compound can be used therapeutically, that is, against already established infections. Other cyclic amines with activity against influenza are under study (e.g. rimantadine and cyclooctylamine). Relatively few additional chemical antiviral agents that hold promise for clinical utility are known at this time. However, there is considerable interest in the potential use of human interferon and interferon inducers. Human interferon is active in model infections, and it thas been reported to be efficacious against influenza in man. Currently, there are serious technical impediments to its economic production. These problems may be circumvented by the use of inducers, but as yet no compound with the required combination of properties has been found. A few preparations have demonstrated high potency in selected model infections, but they appear too toxic for parenteral use in man. The recent observation that an orally active low molecular weight compound (tilorone) possesses interferon inducing activity may pave the way for further advances in this area. Past experiences in viral chemotherapy and an increasing knowledge of the mechanisms of viral infections should better enable us to chart a more productive future course. The processes of compound selection, choice of screening strategy, and market goals can now be better directed. Also, contrary to past predictions, the possibility of therapy as opposed to prophylaxis appears more likely. The present status of viral chemotherapy may be likened to that of the early sulphonamide era in bacteriology. Clinically active compounds are available and the probability of finding more effective agents is very high.

The specificity of antibodies binding 3H-reovirus double-stranded RNA has been studied in an inhibition assay. The antibodies were from human patients with systemic lupus erythematosus, from NZB NZW F 1 mice with a lupus-like syndrome and from young NZB >NZW F 1 mice immunized with RNA in adjuvants. The binding of 14C-polyinosinic polycytidylic acid by selected sera was also studied. The results support the hypothesis that anti-RNA antibodies in lupus may be induced by viral nucleic acid.